A perfusion-based three-dimensional cell culture system to model alveolar rhabdomyosarcoma pathological features

Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system.


Results
Static and perfusion culture of ARMS cells. We first aimed at establishing a 3D culture model enabling a temporally stable, spatially uniform distribution of ARMS cells. In our previous work, the matrisome results highlighted that collagen is one of the main ECM proteins of RMS 21 . Therefore, we decided to use Ultrafoam, a collagen-based commercial scaffold, and compared RMS cell distribution following static or perfusion seeding and culture. We seeded the cells in Ultrafoam using the same cell number in static and perfusion conditions (Fig. 1A,D), and assessed the constructs at 3 time points, up to 15 days. As demonstrated in Fig. 1B-F, the cell distribution in the two conditions was profoundly different. Observing the H&E staining, it is visible how the cells in the static condition were present mainly at the periphery of the scaffold at all time points (Fig. 1B), as also quantified in the graph of Fig. 1C. However, when the static seeding was compared with the perfusion one, the cells were more equally distributed (Fig. 1E,F).
Cell proliferation and pathological ARMS features are enhanced in perfusion culture. We next assessed if the culture system affected the cell proliferation and survival. We performed immunofluorescence for the nuclear proliferation protein KI67 and the apoptotic cleaved caspase 3 (cCas3) marker in our samples and also in xenogeneic tissue samples, as reference. The cell proliferation in the xenogeneic tissue was around 40% while the cell death was close to zero (supplementary Fig. 1). Cells cultured under perfusion flow showed a higher proliferation rate compared to static culture ( Fig. 2A, B). In addition, in both conditions, a low percentage of cells died ( Fig. 2A,B), although the antiapoptotic protein HSP90 (heat shock protein 90) was more expressed in the bioreactor culture compared to the static condition (Fig. 2C).
We interrogated a gene expression dataset for HSP90, comparing its expression in localized versus metastatic ARMS. Interestingly, patients with metastatic disease at diagnosis showed a significantly higher expression of HSP90 than patients with localized disease (Mann-Whitney test P < 0.05, Fig. 2D, and supplementary Fig. 2).
We then aimed at determining if there was a correlation between ECM composition and invasiveness of the RMS. We analyzed publicly available gene expression datasets (GSE108022) comparing the expression of "Matrisome Core Genes" 36 across 3 different groups of patients: Healthy muscle (used as control), ERMS patients, and ARMS patients. The differential expression of Matrisome core genes was able to cluster patients in the defined groups (Healthy, ARMS, and ERMS) (Fig. 3A). Among differentially expressed genes, LAMA1 was over-expressed in patients with ARMS when compared to the healthy control group and ERMS patients.
Conversely, the LAMA2 gene was downregulated in ARMS patients compared with the other two groups. For LAMA1 and LAMA2, box plots and Wilcox-test across the disease groups were also generated (supplementary Fig. 3).
Interestingly, this switch between the expression of LAMA2 to LAMA1 is also reported in RH30 cells cultured in the bioreactor (Fig. 3B-D). In the static condition expression of LAMA2 is higher than in the perfusion www.nature.com/scientificreports/ condition. At the same time, LAMA1 showed significant overexpression when RH30 cells were cultured in the bioreactor both at mRNA and protein level. We also analyzed in our samples the Insulin-like growth factor-binding protein 2 (IGFBP2), whose expression correlates with negative outcomes 37,38 , and we did see a behavior corresponding with patient data (supplementary Fig. 4).
In summary, the cells cultured in the bioreactor expressed the ECM basal lamina LAMA2 and LAMA1 according to the trend found in ARMS patients.
Besides laminins, major elements for cell invasion, we investigated whether other important dissemination factors were present. MMP-2 is an enzyme strictly correlated with ECM remodeling and cell migration. The real-time PCR striking underlined the significantly higher expression in perfusion ARMS samples with respect to the static ones (Fig. 4A). In addition, the MMP-2 activity detected by zymography evidenced the higher presence of the active form in the samples cultured under perfusion (Fig. 4B. Supplementary Fig. 6 for original gel of zymography). Also the metastatic gene CXCR4 was significantly expressed in perfusion condition (supplementary Fig. 7). The RPPA technique confirmed the invasion signature in the same samples. The growth/ dissemination pathway starting from ITGB1, the focal adhesion kinase (FAK) protein, through CDC42, neural Wiskott-Aldrich syndrome protein (n-WASP), and ARP2 demonstrated to be significantly active with respect to the static cultures (Fig. 4C). FAK expression was also analyzed at the different time points in paraffin section of

Discussion
This work establishes an advanced 3D organotypic ARMS model that captures the interaction between cancer cells and the ECM. In this context, we demonstrated that the perfusion flow is paramount to enhance ARMS cell proliferation and expansion. www.nature.com/scientificreports/ Following our proteomic results 21 , the commercially available Ultrafoam ensured unchanged collagen that, together with the flow perfusion, allowed the cells to colonize the scaffold evenly. Ultrafoam mimics rhabdomyosarcoma collagen composition. Collagen type I is the body's most abundant form of collagen (up to 90% of all collagens). It is used in various 3D culture systems, including hydrogels, scaffolds, and spheroids, and it is frequently employed in tissue engineering, drug discovery, and basic research applications. Primary cells or cell lines have already been cultured on Ultrafoam in the perfusion-based bioreactor to create healthy and cancerous tissue models [30][31][32][33]39,40 . Pasini and colleagues 41 already demonstrated how perfusion flow improves migratory phenotype using one breast cancer cell line. www.nature.com/scientificreports/ After the scaffold, the choice of the bioreactor that applies direct flow perfusion was suggested by the promising results already obtained for different tumor cells 32 . Other hydroperfusion conditions, such as in microfluidic platforms, would create fluid flow mainly around the cells and originate inhomogeneous internal transport of nutrients, critically influencing cell behavior 42 . Our system enhances mass transport and flow-induced mechanical shear in a more consistent manner.
We previously developed the automated perfusion bioreactor for the cell seeding of 3D scaffolds, designed to induce continuous oscillatory fluid flow through the scaffold pores. We demonstrated that cell seeding using this device, compared to conventional static or spinner flask techniques, promotes the most efficient cell utilization and uniform cell distribution 43 . Mathematical modeling suggested that spatial variations in cell densities lead to spatial variations in nutrient and metabolic product concentrations within 3D constructs 44 , such that the resulting constructs may have a greater potential to generate uniform 3D tissues. Perfusion of oxygen and nutrients directly through the pores is more effective in maintaining cell viability than the diffusive transport in static seeding/culture. The perfusion method of culture, with the appropriate medium we used, has been effective to grow rhabdomyosarcoma cells.
Indeed, it was striking that both the cell distribution and proliferation in the perfusion condition with respect to the static one, were significantly different, indicating that the cells in the bioreactor were in a comfortable environment.
After validating the operating mode of the model, we started investigating the tumor marker expression. The antiapoptotic gene HSP90, relevant for RMS growth and survival 45 , was significantly expressed in the database of patients with metastatic ARMS. Furthermore, by using the metastatic cell line RH30 cultured in our perfusion model, we also showed high protein expression of this marker. Therefore, the perfusion flow is necessary to mimic metastatic ARMS in vitro.
Using the systematic database approach, we uncovered the different presence of some matrisome genes between ARMS and ERMS, including laminins. In tumors, laminins promote dissemination, increasing proliferation and counteracting apoptosis 46,47 . Strikingly, the proliferating cells cultured in the bioreactor followed the pattern of up and down-regulation of LAMA2 and A1, detected both at the gene level in the patient data set and at the protein level in the Ultrafoam tissue sections. In the perfusion system, the switch of LAMA2, LAMA1 and the enhanced FAK expression underlined the enhanced cell adhesion behavior. In ARMS, which is a musclederived tumor, the downregulation of LAMA2, a major component of the extracellular matrix in skeletal muscle, may contribute to the ability of tumor cells to invade and metastasize to other tissues 48 . In contrast, LAMA1 expression has been found to be upregulated in ARMS. LAMA1 is involved in a variety of cellular processes, including cell migration and angiogenesis, and its upregulation in ARMS may contribute to the ability of tumor cells to migrate and invade surrounding tissues 48 . www.nature.com/scientificreports/ In parallel, there is some evidence to suggest that the proteins CDC42 and ARP2, which are both involved in regulating the actin cytoskeleton and cell migration, may play a role in the switch of LAMA2/LAMA1 expression in the context of ARMS 27 . It has been demonstrated that the upregulation of LAMA1 expression in ARMS was associated with increased activation of the Rho GTPase pathway, which includes CDC42, and with increased expression of ARP2. The researchers demonstrated that inhibition of the Rho/ROCK pathway, which includes CDC42 and other Rho GTPases, led to a decrease in LAMA1 expression and reduced ARMS cell migration and invasion 48 .
These premises allowed us to postulate that the changes of the above mentioned proteins are correlated to migration, typical processes of ARMS cells fusion protein PAX3-FOXO1.
In summary, the switch of LAMA2 and LAMA1 is present in perfusion and not in static; in the perfusion system the ARMS cells PAX3-FOXO1 fusion positive express invasion proteins in a more significant manner with respect to the static culture. Then we can say that in our perfusion system there is a correlation between LAMA2, LAMA1, CDC42, ARP2 expression and cell migration.
We could also find correspondence in our model for IGBP2 but not for the other matrisome genes; we think this aspect could be deeply investigated with primary cells. Despite this, we can say that for LAMA2 and LAMA1 expression, we were able to validate our model. Moreover, laminin enhances the ECM remodeling activity of the gelatinase MMP-2 49 , a protein strongly expressed in ARMS 50 .
RPPA technique allowed us to quantify the expression of the Rho pathway's proteins. It was pretty interesting to observe how the cells cultured in Ultrafoam, both in static and perfusion conditions, expressed ITGA1 indicating an integrin-dependent mechanism of cell migration. Notably, our previous work demonstrated how hyaluronic-based hydrogel induced an integrin-independent mechanism of cell migration, probably due to the CD44-hyaluronan interactions 21 . Along with all the analyzed proteins deputed to cell migration, FAK, CDC42, n-WASP, and ARP2, it was evident how the cells cultured in the bioreactor significantly expressed at a higher level of all the markers with respect to the static condition. However, the precise mechanisms by which these proteins regulate LAMA2/LAMA1 expression and tumor cell behavior are not fully understood and may involve multiple other signaling pathways and factors 48 .
The high expression of the active form ARP2 proved the increased cell motility in the bioreactor 51 . This indicates that the perfusion flow stimulated the cells to proliferate and activate signals relevant to cell growth, potentially prone to dissemination. To study the acquired capacity of our organotypic model to metastasize to a distant site, without altering the microenvironment, we plan to design a new bioreactor system where the ARMS cells can migrate to a recipient tissue.
In summary, our work validates the importance of perfusion-based culture to obtain a 3D organotypic in vitro model of ARMS maintaining uniform cell distribution and active cell proliferation with molecular hallmarks.
We could not use primary patient cells, but the lack of cell variability that characterizes the established cell line was overcome by the fact that ARMS patient specimens possess very low cell population heterogeneity, and cancer cells are the most represented 25 . One limitation of this original work is the lack of functional assays that in the bioreactor could be a drug testing. However, RPPA technique is very sensitive and specific, and we were able to detect increased expression of all the proteins devoted to cell migration such as CDC42, nWASP and ARP2. Future developments of the model with drug testing will be addressed in order to detect the behavior of the primary cells in perfusion conditions.
This three-dimensional model can be helpful in developing new drugs that reach new therapeutic targets.

Methods
Cell lines. ARMS RH30 cell line 52 was kindly provided by the Solid Tumors lab (Prof. Bisogno, Padova, Italy).

ARMS xenografts.
The experimental protocol for the animal care and use was approved by the OPBA (Organismo Per il Benessere degli Animali) local committee (OPBA, protocol 304/2017) and the Ministry of Health under the Italian Law (DL n. 16/92 art. 5). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. In detail, five 12-week-old males and five females Rag2 −/− γc −/− were used as recipients for flank subcutaneous injections and xenograft production. RH30 cells were detached from plastic tissue culture flasks with Dissociation Buffer (Gibco), and suspensions with 2 × 10 6 cells were prepared in 30 mL 1X PBS (Gibco). Xenogeneic ARMS were harvested 21 days post-injection. No control group was required because the aim of the protocol was to achieve the production of the xenogeneic mass. Five mice were injected and all the animals were included. Samples were fixed in PFA 4% and frozen using the cryo-embedding matrix OCT (Fisher Scientific). Cell death and cell proliferation were analyzed by immunofluorescence. Ten images per each sample were acquired with Leica B5000 inverted microscope (20 × magnification). Percentage of positive cells/field was evaluated (positive cells/total nuclei*100). The cell counting was performed in blind by two people. For xenogeneic samples characterization see our published works 21,25 . www.nature.com/scientificreports/ 3D RMS model. Static seeding and culture. Scaffold disks (8 mm diameter × 3 mm height), made from a porous water-insoluble partial hydrochloric acid salt of purified bovine corium collagen sponge, known as Ultrafoam Collagen Hemostat (BD Bard, art # 1,050,050), were soaked in culture medium supplemented with 20% FBS for 1 h at 37 °C. The prepared matrices were placed with sterile tweezers in a 48-well plate. RH30 cell suspension (2 × 10 6 /100ul) was seeded on top of the matrices and placed in the incubator for 1 h at 37 °C; then, 1 ml of culture medium was added.
Perfusion-based seeding and culture. For perfusion 3D culture, we used the U-CUP bioreactor system (Cellec Biotek AG, art # USK001). The Ultrafoam scaffold disks, prepared as described before, were placed between the U-CUP silicon adaptors (Cellec Biotek AG, art # URD08H04) as per manufacturer instruction.
The bioreactors were loaded with 6 mL of media injected from the lower valve. Cells were detached with trypsin 0.05%, counted, and resuspended at concentration 1 × 10 6 /mL, and 2 mL were injected in the bioreactor from the upper valve. The bioreactor was placed in the incubator at 37 °C, 5% CO 2 , and 95% humidity and connected to the syringe pump. Cells were seeded and perfused overnight at a superficial velocity of 400 µm/s. After 24 h of cell-seeding phase, superficial velocity was reduced to 100 µm/s. The media was changed after 3 days, and the culture was conducted for 4, 7, and 15 days.
Cell distribution analysis. Image analysis was performed using Fiji software. Images of the whole scaffold stained with Hematoxylin and Eosin (H&E, Bio-optica) were acquired with Olympus IX71 microscope (20X magnification). Each image has been divided in 5 sections along the direction of the perfusion. Sections were enumerated from 1 (starting from the top of the scaffold) to 5 (including the bottom of the scaffold). N = 3 scaffolds for each condition have been analyzed. Color deconvolution was applied to separate the nuclei from the extracellular matrix. The percentage of nuclei occupancy was calculated as a ratio between Hematoxylin channel over Eosin Channel. The ratio was calculated for each area and normalized over the total Hematoxylin area.
Immunofluorescence. Samples were fixed in 4% PFA for 1 h and dehydrated in sucrose (Sigma-Aldrich) gradients (10%, 15%, 30%). They were finally included in OCT embedding medium (Kaltek) using isopentane (Sigma-Aldrich) fumes chilled on liquid nitrogen. Samples were stored at -80 °C until they were cut in 10 μm slices using Leica CM1520 cryostat (Leica Biosystems). For immunofluorescence analyses, fixed cells or frozen sections were permeabilized for 15 min with 0.5% Triton X-100 (Bio-Rad), blocked for 15 min with 10% horse serum (Gibco) and incubated with primary antibodies overnight at 4 °C. Slides were incubated for 1 h at room temperature with secondary antibodies Alexa Fluor-conjugated protecting them from light. The antibodies used are listed in Table 1. Nuclei were counterstained with 4' ,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) on glass slides or with 1:10.000 Hoechst solution (Sigma-Aldrich) on multiwell plates. Images were acquired with Leica B5000 inverted microscope.
Reverse phase protein array. Reverse Phase Protein Assay (RPPA) was performed as previously described 53,54 . Briefly, ARMS fresh tissue and cell pellets lysed in an appropriate lysis buffer with protease and phosphatase inhibitor were quantified and printed in 4-point dilution curves in quadruplicate on nitrocellulose-coated glass slides (ONCYTER Nitrocellulose Film Slides, Grace BioLabs) with the 2470 Arrayer (Aushon BioSystems). Slides were stained with primary antibodies ( www.nature.com/scientificreports/ amplification stage was performed through the Amplification System Kit and then the signal was revealed using diaminobenzidine/hydrogen peroxide (DAB) as a chromogen-substrate for 5 min (Dako-Cytomation). One slide was stained for the total GAPDH protein amount to normalize the signal intensity of the other antibodies. TIF images of antibodies and GAPDH were analysed using the Microvigene software (VigeneTech Inc, Massachusetts, USA) to extract numeric intensity protein values from the array images.
In total, 123 patients were analyzed; 90 out of 123 showed a local disease, whereas 33 out of 123 possessed a metastatic disease at diagnosis.
Matrisome core genes in RMS. The dataset analyzed during the current study is available using the following accession number n° GSE108022, with RNA-seq data from 101 RMS patients and 5 healthy donors, downloaded from NBC Gene Expression Omnibus (www. ncbi. nlm. nih. gov), was selected as training set for unsupervised cluster analysis due to the inclusion of healthy controls and the high number of patients. Supervised analysis of RNA-seq data was conducted considering only the genes belonging to the "Matrisome Core Genes" category -according to A. Naba's previous publication 36 . These genes were used to perform cluster analysis to discriminate the patients according to their disease group (ARMS, ERMS, or Healthy muscle). A list of the top 50 genes, selected according to their ability to separate the disease groups, was used to repeat the cluster analysis. The analysis was run in "R" software (Author of R software: Core Team (2022). Title: A language and environment for statistical computing. Organisation: Foundation for Statistical Computing, Vienna, Austria. URL https:// www.R-proje ct. org/), in collaboration with Bioinformatic Core Service at IRP "Città della Speranza". Expression levels are normalized among patients: the total expression level of each patient was adjusted to the mean total expression level of the cohort; consequently, expression level of each gene was adjusted with a correcting factor. This is to correct patient-to-patient differences in total mRNA hybridization efficiency on the microarray platform. Data were visualized with a heatmap using the 'heatmap3' function from the 'heatmap3' R package with the 'scale' parameter set to 'row' and the 'balanceColor' parameter set to 'TRUE' .
Zymography. Cells were seeded in a 6-well plate in 1,5 mL serum-free DMEM. The serum-free conditioned medium was harvested after 24 h for zymography. Similarly, the culture medium in the bioreactor was replaced with 6 ml of serum-free medium for 24 h and then collected for zymography. Zymography was carried out as described by Frankowski and colleagues 56 . Briefly, 1% gelatine (J.T. Baker) was added to the 12.5% polyacrylamide gel (Bio-Rad). After the development, the gel was washed in 2.5% Triton X-100 (Bio-Rad) for 1 h and then incubated in a development buffer containing 100 mM CaCl2 and 0.2% NaN3 (Carlo Erba Reagents) overnight. Finally, it was stained in a Coomassie brilliant blue R-250 solution (Bio-Rad, art). Zymography band quantification was performed using Gel Analyzer function in Fiji software 57 .
Real-time PCR. Total RNA was extracted using RNeasy Plus Mini kit (Qiagen) following the supplier's instructions. RNA was quantified with a NanoDrop-2000 spectrophotometer. For all the samples, 0,5 µg of total RNA was reverse transcribed with MultiScribe Reverse Transcriptase (Invitrogen) in a 10 µL reaction containing: 2 ml Buffer 10x (Applied Biosystems), 0,8 ml dNTPs (Applied Biosystems), 2 ml of random primers (Applied Biosystems), 1 ml RNase OUT (Invitrogen) and 3,2 ml RNase free water (Qiagen). Real-Time PCR reactions were performed using a Roche LightCycler II real-time PCR (Roche). Reactions were carried out in duplicate using Sybr Green master mix (Applied Biosystems) and primer mix (final concentration, 200 nM) in a final reaction volume of 15 µL containing: 10 ml Sybr mix (Invitrogen), 2 ml primers, 1 ml BSA (Invitrogen) and 2 ml RNase free water (Qiagen). Relative quantifications (RQ) were calculated by DDCt methods. GAPDH was used as the reference gene for normalization. Primer sequences used are listed in Table 2.